It is currently believed that the release of Ca2+ from the sarcoplasmic reticulum (SR) in skeletal muscle fibers is triggered in responses to depolarization sensed by specialized voltage sensors located in the transverse tubular system (t-system) membranes. The main goal of this application is to understand the dynamic properties of the voltage dependent signal transduction process involving the junction between T-system and the SR. They propose to investigate this process on the same millisecond time scale as the physiological process of T-tubule action potential-elicited Ca2+ release. In order to do so it will be necessary to advance the standard voltage-clamp methodology using a newly developed methodology, supercharging command pulses, that boosts the rate of depolarization in the T-system. T-tubule depolarization will be measured with potentiometric dyes to determine the optimal parameters for supercharging waveforms such that they yield quasi-step depolarizations of the T-system. Ca2+ release from the SR will be measured with a low affinity indicator able to track the rapid kinetics of the release process. The voltage dependence of the transduction process in response to fast T-tubule depolarizations will be investigated in edge segments of muscle fibers with epifluorescence illumination and within localized regions of individual sarcomeres using the confocal spot detection methodology. They will also investigate the mechanisms of stochastic recruitment of Ca2+ release channels by analyzing elementary fluorescence fluctuations related to the opening of RyR channels in both mature muscle fibers and in lipid bilayers. The experimental results are expected to provide new data about the true dynamics and voltage-sensitivity of the Ca2+ release process, leading to a clarified picture of physiological excitation-contraction coupling in skeletal muscle. Since this application deals with general questions regarding voltage propagation in T-tubules, intracellular Ca2+ regulation, voltage dependence of SR Ca2+ release, and fluorescence fluctuations associated with elementary channel openings, its conclusions apply not only to skeletal muscle, but to cardiac muscle, smooth muscle, and to almost every other cell in which Ca2+ regulation processes operate as well.